This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Neutralizing antibodies in HIV-1 infection are directed against the envelope (Env) glycoproteins gp120 and gp41. However, HIV-1 Env utilizes highly effective, but poorly defined, mechanisms to evade antibody-mediated neutralization. The objective of these studies continues to be identifying B cell targets and defining mechanisms of virus neutralization and escape in newly infected subtype C patients using a unique set of reagents derived from newly subtype C HIV-1 infected subjects in a Zambian cohort. In year 02 of this R01, we have demonstrated that escape from autologous neutralization requires multiple mechanisms in early subtype C HIV-1 infection. We have demonstrated complex interactions between multiple regions of Env that reflect the remarkable flexibility of this protein. We have gone on to show that even when an escape mutation creates a potential N-linked glycosylation motif, escape does not necessarily require the motif and can be conferred by other sequence changes. This finding has broad implications for our interpretation of sequence changes that alter potential N-linked glycan motifs. We have shown that V1V2 is highly immunogenic in some patients but to a lesser extent in others. This line of investigation takes on a new level of importance given the renewed interest in quarternary epitopes comprised of V1V2 and other hypervariable domains. With the use of autologous monoclonal antibodies, we have shown escape at the single antibody level, and illustrated how HIV-1 uses a single mutation to simultaneously escape from multiple antibody specificities. In this past year, we have extended these studies to include subtype A infection, covering the two most globally predominant subtypes. These studies will facilitate the identification of similarities and potential differences in Env immunogenicity, predominant Nab targets, and escape mechanisms across subtypes.